Degradation of side chain in sapogenins

ABSTRACT

WHEREIN X is oxo, lower alkylenedioxy, di(lower alkoxy), or IN WHICH R&#39;&#39; is hydrogen or acyl; one of Z and Z&#39;&#39; is oxo or IN WHICH R is hydrogen or acyl, and the other is AND THE DOTTED LINE REPRESENTS THE PRESENCE OR ABSENCE OF A DOUBLE BOND ARE USEFUL AS INTERMEDIATES FOR PREPARING A WIDE VARIETY OF ANDROSTRANES AND ESTRANES, INCLUDING TESTOSTERONE, ESTERONE, ESTRADIOL, ETC., IN PER SE KNOWN MANNER.   Compounds of the formula

United States Patent Kondo et al.

[ 51 May 23, 1972 DEGRADATION OF SIDE CHAIN IN SAPOGENINS Inventors:Eiji Kondo, Ikeda-shi, Osaka; Takashi Mitsugi, Senboku-gun, Osaka, bothof Japan Assignee: Shionogi & Co., Ltd., Osaka, Japan Filed: Sept. 2,1969 Appl. No.: 854,769

Related US. Application Data Continuationdn-part of Ser. No. 597,216,Nov. 28, 1966, abandoned.

US. Cl. ..260/397.3, 260/239.55 C, 260/397.4,

Int. Cl Field of Search References Cited UNITED STATES PATENTSMatsushima 195/51 Primary ExaminerElbert L. Roberts Attorney-Wenderoth,Lind & Ponack ABSTRACT Compounds of the formula wherein X is 0x0, loweralkylenedioxy, di(lower alkoxy), or OR' in which R is hydrogen or acyl;one of Z and Z OR is 0x0 0r in which R is hydrogen or acyl, and theother and the dotted line represents the presence or 18 Claims, NoDrawings DEGRADATION OF SIDE CHAIN IN SAPOGENINS This application is acontinuation-in-part of our application Ser. No. 597,216, filed Nov. 28,I966 now abandoned.

The present invention relates to the degradation reaction of the sidechain of steroidal sapogenins. More particularly, it relates to amicrobiological transformation of spirostane series steroidal sapogeninsinto androstane compounds.

Several investigations dealing with the microbiological transformationof spirostane series steroidal sapogenins have been reported. However,the practical utilization of microorganisms in the direct degradation ofthe spirostane side chain to produce the corresponding androstanecompounds has not been reported so far. The present invention provides,for the first time, a successful microbiological degradation ofspirostane series steroidal sapogenins utilizing Fusarium fungi and theproducts, novel l6-oxygenated androstane compounds useful asinterrnediates for steroidal hormones.

It is a general object of the present invention to provide a newmicrobiological transformau'on of spirostane series sapogenins. It isanother object to provide a new route for production of the androstaneseries steroidal intermediates, from the spirostane series sapogenins.Another object is to provide an industrially available excellentproduction method of androstane series intermediates from naturallyoccurring abundant steroidal materials. Another object is to utilize anovel microbiological transformation in the steroidal partial synthesis.Another object is to provide a new high yield process for the conversionof spirostanes to androstanes consisting of a single microbiologicaltreatment. Another object is to provide novel androstane compounds. Theother objects will be apparent from the following disclosures.

The process of the present invention can illustratively be representedby the following reaction scheme represented by the partial formulasincluding isomers at C C and C and excluding A-C rings of steroidalnucleus, wherein R is a hydrogen atom or OR group, R is a hydrogen atomor an enzymatically acceptable acyl group, and X is group; the saidenzymatically acceptable acyl includes lower alkoxycarbonyl groups andlower aliphatic carboxylic acyl groups:

The starting materials of the present invention are the steroidcompounds having partial formula either I or II. The steroid nucleus ofthe starting materials may possess additional substituent (s) on A, B orC ring, for example, hydroxyl group (5), acyloxy groups (5), 0x0 group(5), unsaturation (s), etc. Especially, the following compounds can beutilized as the starting materials in the present invention:Agapanthagenin,

dehydrogecogenin, 9-dehydromanogenin, diosgenin, diosgenone, gitegenin,hecogenin, isocholegenin, isorhodeasapogenin, jimogenin, kammogenin,kitigenin,

kogagenin, kryptogenin, dihydrokryptogenin, lilagenin, lubigenin,magogenin, manogenin, marcogenin, metagenin, metagenone, mexogenin,neobotogenin, neochlorogenin, neohecogenin, neoruscogenin, neotigogenin,nogiragenin, rhodeasapogenin, ricogenin, rockogenin, ruscogenin,samogenin, sarsasapogenin, sisalagenin, smilagenin, tigogenin,anhydrodehydro-A3,S-tigogenin, tokorogenin, willagenin, yamogenin,yonogenin, yuccagenin and their dehydro and dihydro derivatives and theenzymatically acceptable acyl esters thereof, which are summarized bythe following general formula indicating partial structure inclusive ofrings A,

B and C:

1: u I r C l Ya v wherein Y each represents a hydrogen atom, a hydroiqlgroup or an enzymatically acceptable acyloxy group or, excluding Y and Yrepresents an oxo group, and when Y each is a hydrogen atom, a hydroxylgroup or an enzymatically acceptable acyloxy group they each may beaccompanied with an unsaturation between the neighboring carbon atom,whereby each configuration of Y is a or 3 except for Y, as

As to the enzymatically acceptable acyl group, for example,methoxycarbonyl, ethoxycarbonyl, fonnyl, acetyl, propionyl or the likecan be illustrated.

Generally, the dehydro derivative among these substrates (especiallyA-3-oxo or A"-3-oxo derivatives) and thecompounds having the partialformula ll are transformed more easily and can be utilized as inducersin order to facilitate the transformation of the other startingmaterials.

As for the fungi belonging to Fusarium genus, more particularly, F.solani, F. avenaceum, F. conglulinans, F. vasl'nfectum, F. carcasicum,and the variations or mutants thereof may preferably be utilized.

ln order to obtain desirable growth of microorganisms belonging toFusarium genus for the process of the present invention, a conventionalnutrient medium is used, which, consists of suitable carbon sources,organic or inorganic nitrogen sources, co-factors and inorganic saltsconventionally used in fermentation techniques. Carbon sources may beglucose, sucrose, dextrins, starch, glycerol etc., organic nitrogensources may be peptone, casein, lact-alubumin hydrolysate (Edamin,Scheffield Enzymatic), casein hydrolysate (NZ amin), yeast, yeastextract (Difco), corn steep liquor and like, and inorganic nitrogensources, ammonium nitrate, ammonium phosphate, ammonium sulfate, sodiumnitrate and the like.

The propagation of the fungus belonging to Fusarium genus is carried outin a suitable nutrient medium by stationary culture, but more preferablyby shaking, stirring or submerged culture with aeration. The conditionof propagation i.e. duration, temperature, pH, composition of nutrientmedium or the like may be selected from the conventional order suitablydepending on the nature and quantities of the starting materials.

After the propagation of the microorganism, the living cells in thenutrient medium may be brought to contact with the starting material,which is added as a solution or suspension in acetone, methanol,ethanol, ethylene glycol, propylene glycol, dimethylformamide or thelike, with or without the presence of detergent or surface active agent,under aerobic condition.

Alternatively, the oxidation reaction can be carried out in anundernutrient or nutrient lack medium. For example, the reaction can becarried out in a suspension of mycelia (including spore; throughout thespecification) in distilled water, common water, a buffer solution,saline water or the like; the mycelia is precultured for suitable periodto propagate, iso lated by centrifugation, filtration with suction orthe like, followed by washing with distilled water, a buffered solution,or the like to remove adhering substances on need.

Instead of the mycelia, a homogenate of the mycelia or theisolatedenzyme, which are obtainable by the conventional method from themycelium, may be utilized in this invention.

It is to be understood that the condition of the process of the presentinvention i.e. duration, temperature, pH, composition of the medium orthe like can be selected to attain the best results depending on thenature and concentration of the starting material and used fungus.Usually, duration for l-7 days, temperature of 20-35 C, pH of 4-8 arepreferably selected. These figures are those of a few instances and doesnot restrict the scope of this invention.

In many cases, the fungus belonging to the Fusarium genus does not onlyoxidatively cleave and degradate the spirostane side chain, but inducesthe other oxidative changes on the A-C rings or substituents therein,for example, hydroxylation, hydrolysis, dehydration, oxidation,dehydrogenation etc. These changes are sometimes more preferable forutilization of products of this invention. Among them, a combination ofoxidation and dehydrogenation or dehydration which conducts the productssummarized by the following general forwherein X is group, Z each is ahydrogen atom or a hydroxy group or, excluding Z represents an oxogroup, and when Z each is a hydrogen atom or a hydroxy group they eachmay be accompanied with an unsaturation between the neighboring carbonatom, whereby each configuration of Z is a or ,8 except for Z as a.Specific examples of the products are 1,4-androstadiene-3,16-dione, 16(aand B)-hydroxy-l ,4-androstadien-3-one, 1 1(1): andfl)-hydroxy-l,4-androstadiene- 3,16-dione, l l, l -dihydroxyl,4-androstadien-3-one, 6- hydroxy-] ,4androstadiene-3 16-dione,7-hydroxy-l ,4-androstadiene-3 l 6-dione, 1 6-hydroxyl,4-androstadiene-3, l 2- dione, l,4-androstadiene-3,6, l 6-trione or thelike.

Following the reaction, the reaction products may be recovered by anyone of the conventional methods in the chemical industries. Forinstance, adsorption with an adsorbent such as alumina, silica gel,infusorial earth (Florisil), active charcoal or the like, followed byelution with a solvent system such as petroleum ether, benzene,chloroform, methanol or the like, or extraction of culture medium ormycelia with organic solvents such as chloroform, methylene chloride,ethylene chloride, acetic esters such as butyl acetate, diethyl ketone,butanol or the like can effectively be utilized. After recovery, theproducts may be isolated by concentration of the extracts to a smallvolume or the dryness. Purification of the crude products may beaccomplished by the conventional techniques such as counter currentdistribution method, chromatography over alumina, silica gel or thelike, or more conveniently by a simple recrystallization in aconventional manner.

The products of the present invention serve as valuable intermediatesfor production of various androstane and estrane series steroidalhormones such as androgens, estrogens, antiandrogens, anti-progestins,anti-estrogens, anti-lipemic agents, anti-DOCA agents or the like (e.g.testosterone, l6-estrone, estriol, estrone, estradiol,1,3,5(l0)-estratriene-3,l6- diol, 3-hydroxy-l ,3,5(l0)-estratrien-l6-one and its ketal acylates, 17-haJo-l ,3,5( 10)-estratriene-3, l 6-diol, 1 7-halo-3- hydroxyll ,3,5( l0)-estratrienl6-one, 4-androstene-3 ,16- dione, 5a-androstane-3,l6-dione, ll,l6-dihydroxyl 6-ethynyl-4-androsten-3-one, l,3,5( l0)-estratrienl6-one, l ,3,5( 10 )-estratrien-l 6-ol, 16,17-epithio-l ,4-androstadien-3- one, etc. For example, 3, l o-dioxoandrostal ,4-dienemay be converted into estrogenic 3-hydroxyestra-l,3,5(10)-trien-l6- oneby action of lithium in the presence of hydrogen donor by the processdescribed in the Journal of the American Chemical Society Vol. 86, page742 (1964). The latter may be converted into antilipemic and estrogenic3-acylate-l6-ketal derivatives as described in the US. Pat. No.3,138,590; or the same compound may be converted into estrogenicl7-halo-3- hydroxy-1,3,5( l0)-estratrienl 6-one and alkyl ethers e.g.U.S. Pat. No. 3,079,408 by halogenation at position 17; or the samecompound may be converted into antilipemic lo oxoestral,3,5( l0)-trieneby the process of U.S. Pat. No. 3,081,316;l6-hydroxyl,4-androstadien-3-one may be converted toantideoxycorticosteronic l6,17-epithio1,4-androstadien-3-one by themethod of U.S. Pat. No. 3,123,599; l1B-hydroxy-l,4- androstadiene-3, l6-dione may be converted into useful 1 1,16-dihydroxy-16a-ethynyl-4-androsten-3-one described in Belgian Pat. No.591,544, through 1 l-hydroxy-4-androstene-3,16- dione by the processshown in Chemistry and Industry, page 300 (1962). Further, the compoundshaving l2-oxygen functions are conveniently derived from naturallyoccurring abundant sapogenins, e.g. hecogenin, by the process of thisinvention and the l2-oxygenated derivatives of this invention or thel2-oxygenated derivatives of the useful compounds described above may beconverted into the l2-deoxygenated compounds by e.g. Wolff-Kishnermethod, Huang Minlon method, Clemensen reduction, etc., by e.g. themethod of Shoppee Chemistry of the Steroids, page 121, Butterworth &Co., Ltd. London (1964), if required after protection of oxygenfunctions other than that locating at position 12 or conversion ofl2-oxygen function to l2-oxo group, etc., and said l2-oxygenatedcompounds are thus useful for synthesis of the useful compounds citedabove. Thus the l2-oxo-l6-estrone may be deoxygenated into l6-estrone,which may be ethynylated to afford hypochloesterolaemic l 6a-ethynyl-3,l 6B-dihydroxyl,3,5( l0)-estratriene disclosed in French Patent No.5099M, Netherland Patent No. 65,00801; ll,l6-dihydroxyandrosta-1,4-diene-3-one may be oxidized with an oxidizing agent e.g.N-bromoacetamide, Oppenauer reaction, by e.g. a method analogous to H.L. Herzog et al: the Journal of the American Chemical Society, Vol. 83,page 4073 (1961); 3,1 l-dihydroxy-l,3,5(10)-estratrien-l6-one may betreated with dehydrating agent to 9(1 l)-ene compounds, followed byhydrogenation to obtain estrogenic l6-estrone by a method analogous toBerney J. Magerlein, John A. Hogg; Journal of the American ChemicalSociety, Vol. 80, page 2,220 (1958), which may be converted intohypochloresterolaemic l6a-ethynyl-3,l6fldihydroxy-l,3,5( l0)-estratrieneby the known method described above.

The invention will be described in further detail in the followingexamples which are presented by way of illustration only and not asindicating the scope of the invention.

EXAMPLE 1 Into a medium consisting of 3.5 percent glucose, 2 percentpeptone, 0.3 percent corn steep liquor (pH 5.6), there is inoculated Fusarium solam' and propagated for 2 days at 26-28 C with shaking to giveseed culture. To a nutrient medium of the same composition (each ml. pereach of twenty 500 ml. -shaking flasks), the Fusarium solani seedculture (2 ml. in every flask) is added and cultured for 24 hours withshaking. Diosgenone mg. in 0.8 ml. methanol per each flask) is added tothe broth and cultured for another 3 days. The reaction mixture isadjusted to pH 3 with SN-hydrochloric acid 5 and extracted with ethylacetate. The resultant extract is dissolved in chloroform andchromatographed over silica gel (30 g.). The eluate of chloroform-ether(50:2) is chromatographed over alumina g.) to give benzene-ether (50:6)fraction, from which 3,16-dioxo-l,4-androstadiene (78.5 mg., 38.0percent) is obtained. Mp. 140-141 C. [M 204.&4 (chloroform). Anal:Calcd. for C I-1 0 C 80.24, H 8.51; Found: C 80.27, H 8.58. IR: 111,730, 1658, 1622, 1603 cm".UV:)\ max 245 mp.(e=l5,400).

From the benzene-ether fraction and benzene fraction of the aluminachromatography, 15 1.8 mg. of the starting material is recovered.

EXAMPLE 2 To a nutrient medium of the same composition as in Example l(20 l.) in a liter jar fermenter, there are added a defoaming agent(Uniol p-2,000, 15 ml.) and 500 ml. of Fusarium solani seed culture,obtained by the method as described in Example 1 and cultured for 24hours at 2728 C under aeration (flow rate 20 l./min., gauge pressure0.6-0.7 kg./cm stirring frequency 250 r.p.m.). Diosgenone (2 g.) inmethanol (260 ml.) is added to the broth and cultured for 3 days withvigorous stirring (400-500 r.p.m.). The reaction mixture is treated asin Example 1, and the resultant extract is chromatographed over silicagel (400 g.). The eluent of chloroform ether (50:1) is chromatographedagain over alumina (75 g.) to give benzene and benzene-ether (50:2)fractions, from which 3,16-dioxo-l,4-androstadiene (846 mg., 61.6percent) is obtained.

From the eluent of benzene-ether (100:25), 16/3-hydroxy-1,4-androstadien-3-one (11 mg.) is obtained. Mp. 201202 C. [01],, l5.8i4(chloroform). Anal: Calcd. for C H O C 79.68, H 9.15; Found: C 79.47, H9.01. IR: v 3390, 1654, 1617,1602 cm EXAMPLE 3 A nutrient medium (100ml.) of the same composition as described in Example 1 is inoculatedwith Fusarium solani and cultured for 24 hours. Diosgenin (10 mg.) isadded to the broth and cultured for 6 days. The reaction mixture istreated in the conventional manner and the resultant extract is purifiedby thin layer chromatography (over Kiesel Gel GF, with ethylacetate-toluene(1:l)) to give 3,l6-dioxo-l,4-androstadiene.

max

EXAMPLE 4 A nutrient medium (100 ml.) of the same composition asdescribed in Example 1 is inoculated with Fusarium solani and culturedfor 24 hours. Kryptogenin (10 mg.) is added to the broth and culturedfor 3 days. The reaction mixture is treated in the conventional mannerand the resultant extract is purified by thin layer chromatography (overKiesel Gel GF, with ethyl acetate-toluene (1:1 to give 3,16-dioxo-1,4-androstadiene (3.5 mg.).

EXAMPLE 6 A'-Diosgenone (10 mg.) is oxidized and isolated by thin layerchromatography as in Example 5 to give 1,4-androstadien-3,16-dione (4.2mg.).

EXAMPLE 7 According to the similar manner as in Example 5, diosgenone(10 mg.) is oxidized with shaking to accumulate the degradation enzymein the mycelia. The resultant mycelia are collected by centrifugationand thoroughly washed with a M/ 15 phosphate buffer solution (pH 7.2).The resultant washed mycelia are again suspended in a buffer solution ofthe same composition. Diosgenone (5 mg.) is added to the suspension andcultured at 2628 C for 2 days with shaking. The resultant extract ischromatographed over a thin layer (Kiesel Gel GF, with ethyl acetatetoluene (1:1) to give 1,4-androstadien-3 1 6-dione.

EXAMPLE 8 A nutrient medium (100 ml.) of the same composition asdescribed in Example 1 is inoculated with F usan'um solam and culturedfor 24 hours. Tigogenone (10 mg.) is added to the broth and cultured for6 days. The reaction mixture is treated in the conventional manner andthe resultant extract is purified by thin layer chromatography (overKiesel Gel GF, with ethyl acetate-toluene (1:1) to give3,l6-dioxo-l,4-androstadiene, accompanied with recovery of the startingmaterial.

EXAMPLE 9 Diosgenone (40 g.) is treated in the similar manner asdescribed in Example 2 in twenty 30 liter jar fermenters, and theresulting extract is chromatographed over silica gel (1.1 kg.). Theeluent of chloroform-ether (93:7) is purified by chromatography overalumina to give 3,16-dioxo-1,4androstadiene 18.3 g., 65.4 percent).

The fraction eluted by the solvent more polar than chloroform-ether(:20) of the above mentioned silica gel chromatography, is repeatedlychromatographed over alumina to give 16B-hydroxy-1,4-androstadien-3-one(1.56 g., 5.6 percent) and l6a-hydroxy-1,4-androstadien-3-one (1.29 g.,4.6 percent).

16a-Hydroxy-1,4-androstadien-3-one: Mp. 186-187 C. [01],, 17.4L,(chloroform). Anal: Calcd. for C H O C 79.68, H 9.15 Found: C 79.97, H9.37. IR: 11 ,3396, 1659, 1619,1602 cm. UV 95% m 245.5 I11}L(F16,000).

EXAMPLE 10 The mycelia obtained by the method as described in Example 7is homogenized to give the homogenate (including spore). Kryprogenone (1mg.) is added into a mixture of 30 ml. of M/ 15 phosphate buffersolution and 10 ml. of the homogenate and incubated at room temperaturefor 1 hour. The mixture is extracted with ethyl acetate and the extractis chromatographed over thin layer of silica gel Kiesel Gel GF) withethyl acetate-toluene system (1:1) to give 3,16-dioxo- 1,4-androstadiene.

EXAMPLE 11 According to the similar procedure to the above Examples 3,4, 5 or 8, A -kryptogenone, A-tigogenone, A -tigogenone, kryptogenone,sarsasapogenin, A""-sarsasapogenone, smilagenin, smilagenone,A-smilagenone, A -smilagenone, yamogenin, yamogenone and A-yamogenoneeach affords 1 ,4-androstadiene-3,16-dione.

EXAMPLE 12 A nutrient medium ml. in each of ninety two flasks) of thesame composition as described in Example 1 is inoculated with Fusariumsolani and cultured for 24 hours. Hecogenin (2.76 g., 30 mg in eachflash) is added to the broth and cultured for 7 days. The reactionproduct is extracted with ethyl acetate, and the resultant extract (4.3g) is repeatedly chromatographed over silica gel g.) to afford1,4-androstadiene-3 ,1 2,1 6-trione l6a-hydroxy-1 ,4-androstadiene- 3,12-di0ne, 12a-hydroxy-l ,4-androstadiene-3, l 6-dione, 16ahydroxy-5a-androstene-3, 12-dione andl2a-hydroxy-5oz-androst-1-ene-3,16-dione.

1,4-Androstadiene-3,l2-16-trione: mp. 233234 C. [04],, l05.7i4(chloroform). UV: A max 244 my. (6 15600). 1R: 11 1740, 1715, 1667,1623, 1606 cm. Anal. Calcd. for C f-1 0: C 76.48, H 7.43; Found: C76.56, H 7.75.

1a-Hydroxy-l,4-androstadiene-3,12-dione: mp. 234235 C. [01],, +68.7i0.6(chloroform). UV: max 244 mp. (e= 15000). IR: u 3334, 1714, 1669, 1624,1609 cm". Anal. Calcd. for C H O C 75.97, H 8.05; Found: C 75.83, H8.27.

16a-Acety1oxyl ,4-androstadiene-3,l 2-dione: mp. 169-l 7 1 C, preparedaccording to the conventional acetylation using acetic anyydride inpyridine from the corresponding 16- hydroxy-l ,4-androstadiene-3,12-dione. [a] "-+-27.6:t0. 6 (chloroform). UV: A 243.5 mp. (F1600). IR:1 1740, 1712, 1664, 1625, 1604, 1253, 1250 cm. Anal. Calcd. for C l-I C73,66 H 7.66; Found: C 73.40, H 7.50.

1Za-Hydroxy-l,4-androstadiene-3,16-dione: mp. 226-227 C. [111 l60.4i1(chloroform). UV: A max 245.5 mp. (F 15600). 1R; p 3386, 1744 1664,1620, 1603 cm. Anal. Calcd. for C, H ,O C 75.97, H 8.05; Found: C 76.24,H 8.18.

Acetate of the above: mp. 106-l08 C. IR: Vmax 1741, 1736, 1712,1659,1622, 1601,1253,1239cm. Anal. Calcd. for C H O C 73.66, H 7.66;Found: C 73.69, H 7.94.

1Za-Hydroxy-Sa-androst-1-ene-3,l6-dione: mp. 240242 C. [01],, -100.4fl.4(chloroform). UV: A max 230.5 mp. (e= 10770). IR: v 3456, 1734, 1664,1647, 1595 cm. Anal.Calcd. for G i-1 0 C 75.46, H 8.67; Found: C 75.71,H 8.86.

Acetate of the above: mp 199200 C. [11],, 36.9il.1 (chloroform). UV:229.5 mp. (v.=l0630). IR: Vmax 1744, 1731, 1689, 1600 cm. Anal.Calcd.for C H O C 73.22, H 8.19; Found: C 72.41, H 8.29.

16a-Hydroxy-5a-androst-l-ene-3,12-dione: mp. 226-228 C. [41],,+l09.7fi.70 (chloroform). UV: A 228.5 mp.(e =1ll60).1R:1/ 3478, 1691,1676, 1606 cm. Anal. Calcd. for G Q-1 0 C 75.46, H 8.67; Found: C 75.58,H 8.67.

Acetate: mp. l99200 C. [011] +77.3fl (chloroform). UV: Am 228.5mp.(e=1ll00). 1R: u 1726, 1704, 1687, 1604, 1256 cm. Anal. Calcd. for CI-1 0 C 73.22, H 8.19; Found: C 73.56, H 8.21

Oxidation of the above 1Za-hydroxy-l,4-androstadiene- 3 l 6-dione orla-hydroxy-l ,4-androstadiene-3 l 2-dione with chromium trioxide inacetone gives 1,4-androstadiene- 3,12,16-trione.

Oxidation of lZa-hydroxy-Sa-androst-l-ene, 3,16-dione or 16a-hydroxy-5a-androst-1-ene-3,12-dione with chromium trioxide in acetonegives a-androst-l-ene-3,12,16-trione: mp. 218219 C. [041 66.5i1.8(chloroform). UV: A mu 228.5. mp. (=l0930). 1R: 1 :718.! 1737, 1714,1669, 1609 cm. Anal. Calcd. for C, H ,,O C 75.97, H 8.12; Found: C76.05, H 8.05.

EXAMPLE 13 According to the similar procedure to the above Example 12,botogenin, 3-dehydrobotogenin (botogenone), A'- botogenone, hecogenone(3-dehydrohecogenin), A hecogenone,A -hecogenone, neobotogenin,neohecogenin, gentrogenin, gentrogenone, ricogenin, sisalagenin andjimogenin each affords l,4-androstadiene-3,12,16-trione.

EXAMPLE 14 According to the similar procedure to the above Examples 3,4, 5 or 8, nogiragenin, nogiragenone (3- dehydronogiragenin),A-nogiragenone, A"-nogiragenone, sarmentogenin, 3-dehydro-sarmentogenin,3-dehydro-A -sarmentogenin and 3-dehydro-A -sarmantogenin each affords ll, l 6-dihydroxy-1,4-androstadien-3-one, l la(and ,B)-hydroxy-l,4-androstadiene-3,16-dione and 1 ,4-androstadiene- 3,11,16-trione, andsarmutogenin affords 1,4-androstadiene- 3,1 1,16-trione.

EXAMPLE 15 According to the similar procedure to the above Examples 3,4, 5 or 8, manogenin, gitogenin, tokorogenin, yonogenin and their3-dehydro derivatives each affords several spots being revealed to thecorresponding androstane products.

EXAMPLE 16 To a solution of 1,4-androstadiene3,16-dione (300 mg.) inmethanol (115 ml.) is added sodium borohydride (58 mg.) and let standfor 50 minutes at 0 C. The reaction mixture is treated in theconventional manner to give the extract (362 mg. which ischromatographed over alumina (15 g.). The fraction eluted with benzeneaffords lfl-hydroxy-lA-androstaclien-B-one (269 mg.) and the fractioneluted with ether affords 16a-hydroxy-1,4-androstadien-3-one mg.).

16B-Hydroxy-1,4-androstadien-3-one: mp. 20l202 C. [04],, l5.h4(chloroform). UV: A 245.5 mg 16000). IR: u 3390, 1654, 1617, 1602 cm".Anal. Calcd. for 0, 1 .0 C 79.68, H 9.15; Found: C 79.47, H 9.01.

16 a-Hydroxy-1,4-androstadien-3-one: mp. l86l87 C. [41] "17.4i4(chloroform). UV: A max 245.5 mp. (F 16000). IR: 11 3396, 1659, 1619,1602 6111. Anal. Calcd. for c,,,H ,,o C 79.68, H 9.15; Found: C 79.97, H9.37.

Each l6-hydroxy product afiords the corresponding acetate by theconventional acetylation method with acetic acid in pyridine.

16,8-Acetate: mp. l00-101 C. (a] -22.4:4 (chloroform). UV: A 245.5mp.(t=l6l00). 1R:v 1733, 1664, 1624, 1604 cm Anal. Calcd. for 0 ,11 ,0 1c7679, H 8.59; Found: C 76.97, H 8.84.

1604-Acetate: IR: v 4 1737, 1666, 1632, 1607 cm".

EXAMPLE 17 To a solution of 1,4-androstadiene-3,l6-dione (50 mg.) inbenzene (5 ml.) and 2-butanol (0.5 ml.), aluminum isopropoxide (32mg) isadded and refluxed for 5 hours. The reaction mixture is chromatographedover alumina (3 g.) to give l6B-hydroxy-1,4-androstadien-3-one (14 mg.)and 16ahydroxy- 1 ,4-androstadien-3-one (9 mg.).

EXAMPLE 18 To a solution of 1,4-androstadiene-3,l6-dione (l g.) inbutanone ethylene ketal, p-toluenesulfonic acid (25 mg.) is added andheated for 22.5 hours with azeotropical distillation. The reactionproduct is treated in the conventional manner to give the correspondingethylene ketal.

Biphenyl l g.) is dissolved in tetrahydrofuran (25 m1.) and added withlithium metal mg), and stirred for 20 minutes under reflux. The coloredsolution is added with diphenylmethane (0.5 m1.) andl,4-androstadiene-3,l6-dione l6-ethylene ketal (prepared above) intetrahydrofuran (10 ml.) is added dropwise and refluxed for 35 minuteswith stirring. The reaction mixture is added with methanol and water,acidified with hydrochloric acid (2 ml.) and let stand for 30 minutes at60 C. The reaction product is extracted with chloroform and purified bychromatography over silica gel (100 g.) to give3-hydroxy-l,3,5(lO)-estratrien-l6-one (l6- estrone) (134.3 mg.). Np.248249 C. [oh -99.51? (ethanol). UV: A 281.5 mp (F2190), 287.5 mg. (e=1920 IR: u 3360,1720, 1608, 1582, 1499 cm. Anal. Calcd. for C H O C79.96, H 8.20; Found: C 79.59, H 8.10.

The product (30 mg.) is acetylated with acetic anhydride in pyridine togive the corresponding 3-acetate (18.7 mg.): mp. l32-133 C. [04 -91 .714(ethanol). IR: u 1760,1734, 1615, 1582, 1496 cm. Anal. Calcd. for C I-10 C 76.89, H 7.74; Found: C 76.60, H 7.70.

EXAMPLE 19 To a solution of 16B-hydroxy-1,4-androstadien-3-one (250 mg.)in dihydropyran (7 m1.), phosphoryl chloride (2 drops) is added and letstand for 1.5 hours to give the corresponding 16- tetrahydropyranylether. The product is treated as the preceding Examples withtetrahydrofuran (5.5 g.), biphenyl (475 mg.), lithium metal (35 mg.) anddiphenylmethane (1 drop), and hydrolyzed with concentrated hydrochloricacid as above to give 3,16,6- dihydroxy-1,3,5(10r-estratriene: mp.230231 C. [6!] +72 (ethanol). IR: v 3528, 3238, 1506 cm. Anal. Calcd.for C H O C 79.39, H 8.88; Found: C 78.73, H 8.74.

The product is oxidized with chromium trioxide in acetone to give3-hydroxy-l,3,5(10)-estratrien-16-one: mp. 248249 C.

EXAMPLE 20 16a-Hydroxy-1,4-androstadien-3-one (100 mg.) is treated inthe same manner as above Example 19 to give 3,1601-dihydroxy-1,3,5(10)-estratriene: mp. 228-229 C. [01],, +80- *-4(ethanol). IR: 1.1 3372, 3125, 1613, 1503 cm. Anal. Calcd. for C H O C79.39, H 8.88, Found: C 77.60, H 8.48.

EXAMPLE 21 A mixture of 3-hydroxy-1,3,5(10)-estratrien-16-one (30 mg.),methanol (1 1.5 ml.) and sodium borohydride (6 mg.) is let stand for 50minutes at C. The reaction product is isolated and purified to give3,16B-dihydroxy-1,3,5( lO)-estratriene (19.8 mg.) and3,l6a-dihydroxy-l,3,5( lO)-es tratriene.

EXAMPLE 22 According to the similar procedure to the above Examples18-21, the following conversions are carried out: 1,4-androstadiene-3 l2, l 6-trione to 3-hydroxy-1,3,5( 10)-estratriene-l2,16-dione,16a-hydroxy-1,4-androstadiene-3,12- dione to3,16a-dihydroxy-1,3,5(10)-estratrien-12-one, 12ahydroxy 1,4-androstadiene-3, 16-dione to 3 l 2a-dihydroxy 1,3,5(l0-estratrien-l6-one, 1,3,5( 10 )-estratriene-3,l2a, 16a-triol and1,3,5(10)-estratriene-3,l2a, 16,8-triol, 11(a orB)-hydroxy-1,4-androstadiene-3,l6-dione to 3,1 l-dihydroxy- 1,3 ,5 l0)-estratrienl 6-one, and l 1,16-dihydroxy- 1 ,4-androstadien-3-one to1,3,5( lO)-estratriene-3,l 1,16-triol.

EXAMPLE 23 To a solution of 3,12,16-trioxoandrosta-l,4-diene (240 mg) inethylene glycol (25 ml) is added p-toluene-sulfonic acid monohydrate (7mg) and the mixture is heated at 70 80 C at 5 mm Hg for one hour, whilethe mixture is concentrated to 70 ml. The reaction mixture isalkalinified with a solution of potassium hydroxide in ethanol andpoured into water. The

crystals separated out is filtrated, dissolved in dichloromethane andwashed with water, dried and evaporated to give3,12,16-trioxoandrosta-l,4-diene-16- ethylene ketal.

The latter compound is dissolved in a mixture of triethylene glycol ml),98.5 percent hydrazine (5 ml) and hydrazine hydrochloride (1.4 g), andheated on a bath at 140 C for one hour and then is added potassiumhydroxide (3 g) and heated further one hour at 190 C and ten minutes at210 C. The reaction mixture is poured into ice-water and extracted withchloroform. The extract solution is washed with water, dried andevaporated. The residue is recrystallized from ether hexane to give3,16-dioxoandrosta-l,4-diene-16-ethylene ketal.

The product is dissolved in 70 percent acetic acid and heated on aboiling water bath for one hour. The reaction mixture is diluted withwater and extracted with chloforom. The extract is washed with 5 percentaqueous solution of sodium carbonate and water, dried and evaporated.The residue is recrystallized from a mixture of acetone and hexane togive 103 mg 3,16-dioxandrosta-l,4-diene, m.p. 140- 141 C.

In a similar manner, 3-hydroxyestra-1,3,5( 10)-triene-l2, l 6- dione isconverted into 3-hydroxyestra-1,3,5(l0)-trien-l6- one, m.p. 248 249 C,which is then treated with acetylene and potassium methylate to give3,16B-dihydroxy-l6aethynylestra-1,3,5(10)-triene.

EXAMPLE 24 To a solution of 95 mg 12-oxo-l ,3,5( lO)-estratriene-3,l6[3-diol in ether (10 ml), is added a solution of diazomethane in dry ether.The solution is evaporated in vacuo, and the residue is dissolved inether and washed with water, dried over anhydrous sodium sulfate andevaporated. The residue is dissolved in a mixture of 98.5 percenthydrazine (3 ml) and hydrazine hydrochloride (0.8 g) in triethyleneglycol (5 ml) and the mixture is heated on a bath at 140 C for two hoursand then is added potassium hydroxide (1.5 g) and heated further onehour at 190 C and 30 minutes at 210 C. The reaction mixture is pouredinto ice-water and extracted with chloroform. The extract solution iswashed with water, dried and evaporated. Recrystallization of theresidue from a mixture of ether and hexane affords 1,3,5(10)-estratriene-3,16,B- diol 3-methy1 ether.

EXAMPLE 25 To a solutionof 3,l2a-dihydroxyestra-l,3,5(10)-trien-16- one100 mg) in acetone (10 ml) is added Jones reagent until the color ofchromium trioxide does not disappear. The mixture is poured into water,and extracted with chloroform. The extract solution is washed withwater, dried and evaporated to give l2,16-dioxo-estra-1,3,5(l0)-trien-3-ol (90 mg).

In a similar manner, 1,3,5(10)-estratriene- 3,16-diol is oxidized withchromium trioxide to obtain 16-oxo-1,3,5(10)-estratrien-3-ol.

EXAMPLE 26 A solution of 3,1l-dihydroxy-l6-oxo-l,3,5(10)-estratriene (50mg) in a mixture of ethanol (4 ml) and 10 percent aqueous solution ofsulfuric acid (2 ml) is refluxed for five hours on a water bath. Thereaction mixture is extracted with methylene chloride. The extractsolution is washed with water, dried and evaporated. The residue isdissolved in ethanol (5 ml) and shaken in the hydrogen atmosphere with asuspension of palladium charcoal in ethanol for five hours. Thesuspension is flltrated and concentrated to dryness. Crystallization ofthe residue from ether and pentane affords 16-oxoestral,3,5(l0)-trien-3-ol, m.p. 141 C.

In a similar manner, 1,3,5(10)-estratriene-3,l 1,16a-triol is convertedinto l,3,5(l0)-estratriene-3,l6a-diol, m.p. 228 229 C.

EXAMPLE 27 To a solution of l l,l6-dihydroxyandrosta-1,4-dien-3-one(0.54 g) in a mixture of acetone (40 m1), methanol (10 ml) and water 10ml) at room temperature is added N- bromoacetamide (1.93 g). Afterstanding for three hours in the dark at 5 10 C, the reaction mixture ispoured into 5 percent aqueous solution of sodium sulfate and the productis extracted with methylene chloride. The dried extract solution isconcentrated and the residual oil is recrystallized from a mixture ofether and hexane to afford 3,16-dioxoandrostal ,4- dien-l 1-01.

The product is dissolved in a mixture of tetrahydrofuran 15 ml) andether 15 ml) and the solution is added dropwise into a solution ofpotassium metal (880 mg) in a mixture of tetrahydrofuran (15 ml) andether 15 ml) saturated with acetylene gas. The mixture is bubbled withacetylene gas for 3 hours and then aqueous ammonium chloride is addedthereto. The reaction mixture is washed successively with water, aqueoussodium carbonate solution and water, dried and evaporated in vacuo.Recrystallization of the residue from a mixture of acetone and pentaneaffords 3,11,16-trihydroxy- 1 6-ethynylandrosta-1,4-diene.

A mixed solution of lithium (60 mg) and biphenyl 1,2 g) intetrahydrofuran (20 ml) is stirred for two hours at room temperature.Into the solution cooled at 65 C is added 3,1 1,16-trihydroxy-l6-ethynylandrosta-l,4-diene obtained above and the mixtureis stirred for 45 minutes at the temperature. Then ammonium chloride (2g) is added thereto, and the temperature is raised gradually to 40 Cduring 2 hours. The reaction mixture is poured into saturated aqueoussolution of ammonium chloride (20 ml) and extracted with methylenechloride. The extract solution is washed with water, dried andevaporated in vacuo. Purification of the residue by chromatography overalumina and recrystallization from a mixture of ethyl acetate and etheraffords 3,1 13,16,8-trihydroxy-l 6aethynylandrost-4-ene.

What we claim is:

1. A compound of the formula wherein X is oxo, lower alkylenedioxy, di(lower alkoxy), or

in which R is hydrogen or acyl; one of Z and Z is oxo or in which R ishydrogen or acyl, and the other is the dotted line represents thepresence or absence of a double bond.

2. A compound according to claim 1, namely, l,4-androstadiene-312,16-trione.

3. A compound according to claim 1, namely, l6a-hydroxyl,4-androstadiene3, l Z-dione.

4. A compound according to claim 1, namely, l2a-hydroxy-1,4-androsladiene-3, l6-dione.

5. A compound according to claim 1, namely, la-hydroxya-androstl -ene-3l Z-dione.

6. A compound according to claim 1, namely, IZa-hydroxy-Sa-androstl-ene-3, l 6-dione.

7. A compound according to claim 1, namely, 1 1 (aor fl)- hydroxy-l,4-androstadiene-3, l 6- dione.

8. A compound according to claim 1, namely, l 1,16- dihydroxy-l,4-androstadien-3 -one.

9. A compound of the formula wherein X 0, a lower alkylenedioxy group,di(lower alkoxy) group or and R is a hydrogen atom or an acyl group.

10. A compound according to claim 9, namely, l,4-androsradiene-3 l6-dione.

11. A compound according to claim 9, namely. I Gar-hydroxyl,4-androstadien-3-one.

12. A compound according to claim 9, namely, l6B-hydrox- 5 yl,4androstadien-3-one.

13. A compound of the formula wherein R is hydrogen, lower alkyl oracyl; X is 0x0 or H one ofZ and Z is 0x0 or and the other is 14. Acompound according to claim 13, namely, 3-hydroxyl ,3,5(l0)-estratriene-l2,16-dione.

15. A compound according to claim 13, namely, 3,16- dihydroxy- 1 ,3 ,5(l0 )-estratrien- 1 2-one.

A compound according to claim 13, namely, 3,120:- dihydroxyl ,3 ,5( l0)-estratrienl 6-one.

17. A compound according to claim 13, namely, 3,11- dihydroxy, l ,3,5(lO)-estratrienl 6-one.

18. A compound according to claim 13, namely, l,3,5( l0)- estratriene-3,l l l6-triol,

2. A compound according to claim 1, namely, 1,4-androstadiene-3, 12,16-trione.
 3. A compound according to claim 1, namely, 16 Alpha -hydroxy-1, 4-androstadiene-3,12-dione.
 4. A compound according to claim 1, namely, 12 Alpha -hydroxy-1, 4-androstadiene-3,16-dione.
 5. A compound according to claim 1, namely, 16 Alpha -hydroxy-5 Alpha -androst-1-ene-3,12-dione.
 6. A compound according to claim 1, namely, 12 Alpha -hydroxy-5 Alpha -androst-1-ene-3,16-dione.
 7. A compound according to claim 1, namely, 11 ( Alpha or Beta )-hydroxy-1,4-androstadiene-3,16- dione.
 8. A compound according to claim 1, namely, 11,16-dihydroxy-1,4-androstadien-3-one.
 9. A compound of the formula
 10. A compound according to claim 9, namely, 1,4-androstadiene-3,16-dione.
 11. A compound according to claim 9, namely, 16 Alpha -hydroxy-1,4-androstadien-3-one.
 12. A compound according to claim 9, namely, 16 Beta -hydroxy-1, 4-androstadien-3-one.
 13. A compound of the formula
 14. A compound according to claim 13, namely, 3-hydroxy-1,3, 5(10)-estratriene-12,16-dione.
 15. A compound according to claim 13, namely, 3,16-dihydroxy-1, 3,5(10)-estratrien-12-one.
 16. A compound according to claim 13, namely, 3,12 Alpha -dihydroxy-1,3,5(10)-estratrien-16-one.
 17. A compound according to claim 13, namely, 3,11-dihydroxy,1, 3,5(10)-estratrien-16-one.
 18. A compound according to claim 13, namely, 1,3,5(10)-estratriene-3,11,16-triol. 